Activity of vincristine, L-ASP, and dexamethasone against acute lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo by Min H. Kang, Yun Hee Kang, Barbara Szymanska, Urszula Wilczynska-Kalak, Michael A. Sheard, Theresa M. Harned, Richard B. Lock, and C. Patrick Reynolds Blood Volume 110(6):2057-2066 September 15, 2007 ©2007 by American Society of Hematology

Combination cytotoxicity of ABT-737 and L-ASP, vincristine, or dexamethasone. Combination cytotoxicity of ABT-737 and L-ASP, vincristine, or dexamethasone. Dose-response curves of ALL cell lines to ABT-737 (●), L-ASP (△), vincristine (VCR; ○), dexamethasone (DEX; □), and the combinations: ABT-737 + L-ASP (▲), ABT-737 + VCR (), and ABT-737 + DEX (gray squares). The concentrations applied for the cell lines were 0.5 to 5 μM for ABT-737; 0.1 to 1 IU/mL for L-ASP; 5 to 50 ng/mL for VCR; and 50 to 500 nM for DEX. Each condition had 12 replicates, and error bars represent standard deviation. The CI values are shown in Table 1. Min H. Kang et al. Blood 2007;110:2057-2066 ©2007 by American Society of Hematology

Activation of various proapoptotic proteins and caspase-8, caspase-9, and caspase-3. Activation of various proapoptotic proteins and caspase-8, caspase-9, and caspase-3. Whole-cell extracts from either COG-LL-317 (left panels) or RS4-11 (right panels) cells were incubated with ABT-737 (2.5 μM), L-ASP (2.5 IU/mL), or the combination over time, after which protein extracts were immunoblotted with the specified antibodies for proapoptotic proteins (A) and caspase-8, caspase-9, and caspase-3 (B). The data shown are representative of 3 experiments. Vertical lines have been inserted to indicate where a gel lane was cut. These gels came from 2 different experiments. Min H. Kang et al. Blood 2007;110:2057-2066 ©2007 by American Society of Hematology

ABT-737 plus L-ASP caused enhanced apoptosis, mitochondrial membrane depolarization, and release of mitochondrial cytochrome c. ABT-737 plus L-ASP caused enhanced apoptosis, mitochondrial membrane depolarization, and release of mitochondrial cytochrome c. Annexin V–FITC and JC-1 assay. COG-LL-317 cells were incubated with ABT-737 (2.5 μM), L-ASP (2.5 IU/mL), or the combination for 6 hours. Then, cells were incubated with annexin V–FITC and PI (A) or JC-1 (B,C) and analyzed by flow cytometry. (A) Bars show the percentage of cells that were annexin V–FITC+/PI−, defined as apoptotic, and error bars represent standard deviation. (B) The loss of Δψm by ABT-737, L-ASP, or ABT-737 + L-ASP was measured by flow cytometry using the JC-1 mitochondrial probe. The bars show the percentage of mitochondrial membrane–depolarized cells. (C) Cytograms showing representative JC-1 assays for mitochondrial membrane potential. The transition of red fluorescence to green indicates mitochondrial membrane depolarization by the drug(s). The values represent the percentage of mitochondrial membrane-depolarized cells. Total number of events analyzed for each condition was 10 000. (D) Cytochrome c release after drug treatment. Cytosolic extracts from COG-LL-317 that had been incubated for 6 hours with ABT-737 (2.5 μM), L-ASP (2.5 IU/mL), or both drugs in combination were prepared and immunoblotted with cytochrome c antibody. The blot shown is representative of 2 separate experiments. Min H. Kang et al. Blood 2007;110:2057-2066 ©2007 by American Society of Hematology

Cytotoxicity of ABT-737 plus vincristine, dexamethasone, or L-ASP in PMBCs. (A) Dose-response curves of PBMCs to ABT-737 (●), L-ASP (△), vincristine (VCR; ○), dexamethasone (DEX; □), and the combinations (gray symbols). Cytotoxicity of ABT-737 plus vincristine, dexamethasone, or L-ASP in PMBCs. (A) Dose-response curves of PBMCs to ABT-737 (●), L-ASP (△), vincristine (VCR; ○), dexamethasone (DEX; □), and the combinations (gray symbols). The concentrations of ASP used for PBMCs were increased to 1 to 10 IU/mL to assure examination of cytotoxicity in clinically achievable concentrations. Each condition had 12 replicates, and error bars represent standard deviation. (B) Cells (COG-LL-317 and PBMCs) were incubated with ABT-737 (2.5 μM), ASP (1 IU/mL), or the combination for 6 hours. Then, apoptosis assessed with annexin V and PI staining measured by flow cytometry. COG-LL-317 cells were used as positive controls. For PBMCs, apoptosis was assessed only in lymphocytes that fell within a standard forward- and side-scatter lymphocyte gate. Cells that were annexin V–FITC+, PI− were defined as apoptotic. The bar graph shows the percentage of cells in apoptosis, and the values represent means (± SD) of 3 samples, and the experiments were repeated twice. Apoptosis in COG-LL-317 for ABT-737 + L-ASP was significantly higher than control (P < .001), while neither single agents nor ABT-737 + L-ASP induced significant apoptosis in lymphocytes. Min H. Kang et al. Blood 2007;110:2057-2066 ©2007 by American Society of Hematology

Four-drug combination cytotoxicity of ABT-737 in combination with VXL in ALL cell lines. Four-drug combination cytotoxicity of ABT-737 in combination with VXL in ALL cell lines. Dose-response curves of ALL cell lines to ABT-737 (●), VXL (△), and the combinations of all 4 drugs (▲). The concentrations for VCR, DEX, and L-ASP applied were 0.5 to 5 ng/mL, 50 to 500 nM, and 0.1 to 1 IU/mL, respectively, where 1 U on the VXL axis corresponds to 1 ng/mL VCR, 100 nM DEX, and 0.2 IU/mL L-ASP. The concentrations for ABT-737 applied were 1 to 10 μM for CEM, COG-LL-317, MOLT-3, MOLT-4, and NALM-6; concentrations of 0.1 to 1 μM were used for RS4-11* and COG-LL-319*. Each condition had 12 replicates, and error bars represent SD. *Cell lines especially sensitive to ABT-737 as a single agent (Figure 1) that required lower dosing for combination drug testing. Min H. Kang et al. Blood 2007;110:2057-2066 ©2007 by American Society of Hematology

In vivo efficacy of ABT-737 combined with a VXL treatment regimen. In vivo efficacy of ABT-737 combined with a VXL treatment regimen. NOD/SCID mice were inoculated with ALL-7 (A) or ALL-19 (B), monitored for engraftment, and treated with vehicle control (Con; thin lines), ABT-737 (737; bold dotted lines), a combination of vincristine, dexamethasone, and L-ASP (VXL; thin dotted lines), or VXL plus ABT-737 (VXL + 737; bold lines). Drugs were administered by intraperitoneal injection: vincristine (Sigma-Aldrich, Castle Hill, Australia), 0.15 mg/kg in saline every 7 days for 4 weeks; dexamethasone (Sigma-Aldrich), 5 mg/kg in saline Monday to Friday for 4 weeks; L-ASP (Aventis, Lane Cove, Australia), 1000 IU/kg in saline Monday to Friday for 4 weeks; and ABT-737, 25 mg/kg in DMSO (final concentration < 1%), 30% propylene glycol, 5% Tween 80, and 65% dextrose (pH 4-5), Monday to Friday for 4 weeks. The EFS of mice was quantified as the time taken from the initiation of treatment until leukemia cells reached 25% in the peripheral blood, or for mice to be killed due to treatment-related toxicity. Each line represents the proportion of mice remaining event-free over time. Min H. Kang et al. Blood 2007;110:2057-2066 ©2007 by American Society of Hematology